20.500.12556/DiRROS-19697 Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA Quantitative PCR (qPCR) is an important tool in pathogen detection. However, the use of different qPCR components, calibration materials and DNA extraction methods reduces comparability between laboratories, which can result in false diagnosis and discrepancies in patient care. The wider establishment of a metrological framework for nucleic acid tests could improve the degree of standardisation of pathogen detection and the quantification methods applied in the clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures, and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) has already been used for pathogen quantification by analysing nucleic acids. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessment of its actual performance characteristics is needed before it can be implemented in a metrological framework, and to allow adequate estimation of measurement uncertainties. Here, four laboratories demonstrated reproducibility (expanded measurement uncertainties below 15%) of dPCR for quantification of DNA from human cytomegalovirus, with no calibration to a common reference material. Using whole-virus material and extracted DNA, an intermediate precision (coefficients of variation below 25%) between three consecutive experiments was noted. Furthermore, discrepancies in estimated mean DNA copy number concentrations between laboratories were less than twofold, with DNA extraction as the main source of variability. These data demonstrate that dPCR offers a repeatable and reproducible method for quantification of viral DNA, and due to its satisfactory performance should be considered as candidate for reference methods for implementation in a metrological framework. digital PCR DNA quantification inter-laboratory assessment human cytomegalovirus virus reference materials true false true Angleški jezik Ni določen Neznano 2024-07-24 03:45:37 2024-07-24 15:07:16 2024-07-25 04:14:55 0000-00-00 00:00:00 2017 0 0 str. 2601-2614 iss. 10 Vol. 409 2017 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 2017-01-26 578 1618-2642 10.1007/s00216-017-0206-0 4223055 http://dx.doi.org/10.1007/s00216-017-0206-0 1 c1112805-494d-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=27229 RAZ_Pavsic_Jernej_2017.pdf RAZ_Pavsic_Jernej_2017.pdf 1 315D9D64042B8F34F5B431556FF38B60 f6681254d4bb77e8e4e25b57209f17b288db674231481f758cb975c21b983336 fb5e351f-49ac-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=27374 Nacionalni inštitut za biologijo 0 0 0